Clinical analyses of DNA sequences are typically directed to determining how a gene in a patient sample differs from a prototypical normal sequence. DNA sequencing through the chain termination method developed by Sanger and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA 1977; 74: 5463-5467), and the chemical degradation method developed by Maxam and Gilbert (Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 1977; 74: 560-564), or using techniques such as Sequencing By Hybridization (SBH) or Sequencing By Synthesis (see e.g. WO 93/21340) all have the potential to identify mutations and in the same process also reveal the consequence of the mutation at the level of protein coding etc.
Another sequencing approach is disclosed in EP-A-223 618 which described the use of an immobilised DNA template, primer and polymerase exposed to a flow containing only one species of deoxynucleotide at a time. A downstream detection system then determines whether deoxynucleotide is incorporated into the copy or not by detecting the difference in deoxynucleotide concentrations entering and leaving the flow cell containing the complex of DNA template and polymerase.
For screening purposes, however, it is often sufficient, at least initially, to identify deviations from the normal sequence but without directly revealing how a sequence differs from the normal one or only roughly locating the mutation. There are a number of such techniques which speed up analysis as compared to those that involve DNA sequence determination.
One such method uses "label-free" detection based on surface plasmon resonance (SPR) for determining the binding of a short oligonucleotide probe to a single-stranded target sequence immobilised to a sensor chip. Since a mismatch significantly affects the binding affinity, the presence of a sequence deviation may be determined. This method has, however, several disadvantages, such as that it requires immobilizing long target sequences, usually PCR products, to the sensor chip and that the sensor chip can not be regenerated.